Schiza Natasa, Sargiannidou Irene, Kagiava Alexia, Karaiskos Christos, Nearchou Marianna, Kleopa Kleopas A
Neuroscience Laboratory.
Department of Molecular Pathology and Electron Microscopy, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.
Hum Mol Genet. 2015 Apr 1;24(7):2049-64. doi: 10.1093/hmg/ddu725. Epub 2014 Dec 18.
Oligodendrocytes are coupled by gap junctions (GJs) formed mainly by connexin47 (Cx47) and Cx32. Recessive GJC2/Cx47 mutations cause Pelizaeus-Merzbacher-like disease, a hypomyelinating leukodystrophy, while GJB1/Cx32 mutations cause neuropathy and chronic or acute-transient encephalopathy syndromes. Cx32/Cx47 double knockout (Cx32/Cx47dKO) mice develop severe CNS demyelination beginning at 1 month of age leading to death within weeks, offering a relevant model to study disease mechanisms. In order to clarify whether the loss of oligodendrocyte connexins has cell autonomous effects, we generated transgenic mice expressing the wild-type human Cx32 under the control of the mouse proteolipid protein promoter, obtaining exogenous hCx32 expression in oligodendrocytes. By crossing these mice with Cx32KO mice, we obtained expression of hCx32 on Cx32KO background. Immunohistochemical and immunoblot analysis confirmed strong CNS expression of hCx32 specifically in oligodendrocytes and correct localization forming GJs at cell bodies and along the myelin sheath. TG(+)Cx32/Cx47dKO mice generated by further crossing with Cx47KO mice showed that transgenic expression of hCx32 rescued the severe early phenotype of CNS demyelination in Cx32/Cx47dKO mice, resulting in marked improvement of behavioral abnormalities at 1 month of age, and preventing the early mortality. Furthermore, TG(+)Cx32/Cx47dKO mice showed significant improvement of myelination compared with Cx32/Cx47dKO CNS at 1 month of age, while the inflammatory and astrogliotic changes were fully reversed. Our study confirms that loss of oligodendrocyte GJs has cell autonomous effects and that re-establishment of GJ connectivity by replacement of least one GJ protein provides correction of the leukodystrophy phenotype.
少突胶质细胞通过主要由连接蛋白47（Cx47）和Cx32形成的缝隙连接（GJ）相互耦联。隐性GJC2/Cx47突变会导致佩利措伊斯-梅茨巴赫样病，一种髓鞘形成不良性脑白质营养不良，而GJB1/Cx32突变会导致神经病变以及慢性或急性短暂性脑病综合征。Cx32/Cx47双敲除（Cx32/Cx47dKO）小鼠从1月龄开始出现严重的中枢神经系统脱髓鞘，数周内导致死亡，为研究疾病机制提供了一个相关模型。为了阐明少突胶质细胞连接蛋白的缺失是否具有细胞自主效应，我们构建了在小鼠蛋白脂蛋白启动子控制下表达野生型人Cx32的转基因小鼠，从而在少突胶质细胞中获得外源性hCx32表达。通过将这些小鼠与Cx32KO小鼠杂交，我们在Cx32KO背景上获得了hCx32的表达。免疫组织化学和免疫印迹分析证实hCx32在中枢神经系统中主要在少突胶质细胞中强烈表达，并且在细胞体和髓鞘上形成GJ的定位正确。通过与Cx47KO小鼠进一步杂交产生的TG(+)Cx32/Cx47dKO小鼠表明，hCx32的转基因表达挽救了Cx32/Cx47dKO小鼠严重的早期中枢神经系统脱髓鞘表型，导致1月龄时行为异常明显改善，并防止了早期死亡。此外，与1月龄的Cx32/Cx47dKO中枢神经系统相比，TG(+)Cx32/Cx47dKO小鼠的髓鞘形成有显著改善，而炎症和星形胶质细胞增生性变化完全逆转。我们的研究证实少突胶质细胞GJ的缺失具有细胞自主效应，并且通过替换至少一种GJ蛋白来重新建立GJ连接性可纠正脑白质营养不良表型。
